Numerous microorganisms have the ability to accumulate intracellular reserves of Poly [(R)-3-hydroxyalkanoates] (PHAs). PHAs are biodegradable and biocompatible thermoplastic materials, produced from renewable resources, with a broad range of industrial and biomedical applications (Williams and Peoples, 1996, CHEMTECH 26, 38-44). The PHA biopolymers encompass a broad class of polyesters with different monomer compositions and a wide range of physical properties. To date around 100 different monomers have been incorporated into the PHA polymers (Steinbüchel and Valentin, 1995, FEMS Microbiol. Lett. 128; 219-228). PHAs can be divided into two groups according to the length of their side chains. Those with short side chains, such as polyhydroxybutyrate (PHB), a homopolymer of R-3-hydroxybutyric acid units, are crystalline thermoplastics, whereas PHAs with medium length side chains, such as polyhydroxyoctanoic or polyhydroxydecanoic acid, are more elastomeric.
In bacteria, each PHA group is produced by a specific pathway. In the case of the short pendant group PHAs, three enzymes are involved, a β-ketothiolase, an acetoacetyl-CoA reductase, and a PHA synthase. For example, in PHB biosynthesis two molecules of acetyl-coenzyme A are condensed by a β-ketothiolase to yield acetoacetyl-coenzyme A. The latter is then reduced to the chiral intermediate R-3-hydroxybutyryl-coenzyme A by the reductase, and subsequently polymerized by the PHA synthase enzyme. Short chain length PHA synthases typically allow polymerization of C3-C5 hydroxy acid monomers including both 4-hydroxy and 5-hydroxy acid units. This biosynthetic pathway is found in a number of bacteria such as Ralstonia eutropha, Alcaligenes latus, Zoogloea ramigera, etc (Madison, L. L. & Huisman, G. W. Microbiology and Molecular Biology Reviews 1999, 63, 21-53).
Medium chain length pendant group PHAs are produced by many different Pseudomonas bacteria. The hydroxyacyl-coenzyme A monomeric units can originate from fatty acid β-oxidation and fatty acid biosynthetic pathways. The monomer units are then converted to polymer by PHA synthases which have substrate specificity's favoring the larger C6-C14 monomeric units (Madison, L. L. & Huisman, G. W. Microbiology and Molecular Biology Reviews 1999, 63, 21-53). In the Pseudomonas organisms, the PHA synthases responsible for production of the long pendant group PHAs were found to be encoded on the pha locus, specifically by the phaA and phaC genes (U.S. Pat. Nos. 5,245,023; 5,250,430; Huisman et. al., 1991, J. Biol. Chem. 266:2191-2198).
Co-polymers comprised of both short and medium chain length pendant groups can also be produced in bacteria possessing a PHA synthase with a broad substrate specificity. For example, Pseudomonas sp. A33 (Appl. Microbiol. Biotechnol. 1995, 42, 901-909), Pseudomonas sp. 61-3 (Kato, M., Bao, H. J., Kang, C. -K, Fukui, T., Doi, Y. Appl. Microbiol. Biotechnol. 1996, 45, 363-370), and Thiocapsa pfennigii (U.S. Pat. No. 6,011,144) all possess PHA synthases that have been reported to produce co-polymers of short and medium chain length monomer units.
An enzyme encoded by phaG was recently identified in both Pseudomonas putida and Pseudomonas aeruginosa and has been reported to be the link between fatty acid biosynthesis and medium chain length PHA formation (see Pathway A in FIG. 1) in these organisms (Rehm, B. H. A., Kruger, N., Steinbuchel, A. J. Biol. Chem. 1998, 273, 24044-24051; WO 98/06854; U.S. Pat. No. 5,750,848; Hoffmann, N., Steinbuchel, A., Rehm, B. H. A. FEMS Microbiology Letters, 2000, 184, 253-259). In these studies, PhaG was identified as a 3-hydroxyacyl-acyl carrier protein-coenzyme A transferase based on the ability of partially purified enzyme preparations to convert 3-hydroxydecanoyl CoA in the presence of ACP to 3-hydroxydecanoyl ACP (Rehm, B. H. A., Kruger, N., Steinbuchel, A. J. Biol. Chem. 1998, 273, 24044-24051). Expression of PhaG and PhaC in Pseudomonas fragi, an organism that does not naturally produce PHAs as a storage material, enabled the production of PHAs from gluconate (Fiedler, S., Steinbuchel, A., Rehm, B. H. A. Applied and Environmental Microbiology 2000, 66, 2117-2124). No polymer however was observed upon expression of a medium chain length synthase and PhaG in E. coli (Rehm, B. H. A., Kruger, N., Steinbuchel, A. J. Biol. Chem. 1998, 273, 24044-24051). While E. coli is capable of producing small amounts of low molecular weight, non-granule forms of PHB (Reusch, R. N. Can. J. Microbiol. 1995, 41 (suppl. 1), 50-54), like P. fragi, it is unable to produce granules of storage polymer.
U.S. Pat. No. 5,750,848 reported that the phaG gene from Pseudomonas putida encodes a 3-hydroxyacyl-ACP—CoA transferase activity useful for producing (D)-3-hydroxyacyl-CoA precursors for the biosynthesis of polyhydroxyalkanoate (PHA) biopolymers comprising C8 and C10 units. This activity has not been confirmed, however.
It is therefore an object of the present invention to express PhaG in conjunction with an acyl CoA synthetase and a PHA synthase in an organism for the production of PHAs.
It is therefore further object of the present invention to express PhaG in conjunction with an acyl CoA synthetase and a PHA synthase in an organism for the production of medium chain length PHAs.